Foot and Mouth Disease (FMD) is an acute systemic viral infection that affects food producing animals, such as cattle, sheep, goats, swine and other cloven-hoofed animals. Despite its very low mortality rate, the highly contagious nature of FMD makes it one of the most serious diseases of the livestock industry in terms of productivity losses and economic impact.
The disease is endemic in many parts of the world. The World Organization for Animal Health (OIE) periodically publishes disease distribution and outbreak maps. The sanitary status granted by the OIE has a profound economic impact in countries with meat trade-dependent economies because of the market restrictions that OIE imposes, especially on countries affected by FMD.
Effective vaccines and stringent control programs have eradicated the disease in most developed countries but, regardless of strict international trade policies, major outbreaks have occurred relatively recently in Europe (2000, 2001) and in Japan (2000, 2010).
Even though many countries have obtained the status of “free from FMD with vaccination” in some or all of their territory, the occurrence of outbreaks in neighboring countries represents a constant threat.
Despite continuous efforts to develop recombinant subunit vaccines that would not require propagation of the pathogen in large scale, current vaccines are based on inactivated whole virus concentrated and purified to reach a critical mass of antigen capable of generating a protective immune response. These vaccines are manufactured in plants with NBS biological safety level 4 (OIE). It is estimated that between 2.5 and 3 billion doses are produced annually worldwide.
Additionally, many countries have established emergency programs that include storage of frozen FMD antigens, called antigens banks, that would enable quick formulation of emergency vaccines in case of an FMDV pandemic.
The efficacy of inactivated virus vaccines, which are routinely used as part of eradication programs and in emergency contexts, is highly dependent on the antigenic payload formulated in each dose and on virus integrity.
The FMD virus (FMDV) is a non-lipid-enveloped virus featuring an icosahedral symmetry and a described size (diameter) ranging from 28 to 40 nm. The whole virus particle is extremely labile in vitro, it dissociates into monomers at temperatures above 56° C. and pH below 6. Seven FMDV serotypes have been reported, designated as O, A, C, SAT1, SAT2, SAT3 and Asia1.
The 140S (146S) quantitative sucrose density gradient analysis is the recommended method to quantify virus antigen and, on that basis, formulate vaccines. The 140S (146S) method, as developed by Barteling and Meloen (Barteling S J, Meloen R H. A simple method for the quantification of 140S particles of foot-and-mouth disease virus (FMDV). Arch Gesamte Virusforsch, 1974; 45(4):362-4), has provided over the past three decades a reliable method for virus concentration measurement. The method consists of ultracentrifugation of the sample in a sucrose concentration gradient from about 20 to 45%. The sucrose gradients are prepared by layering sucrose solutions of decreasing concentration either by hand or using simple gradient mixers.
The principle of the 146S method is to separate the FMD viral particles based on their sedimentation coefficient in the sucrose gradient. Therefore, the technique provides only an indirect measurement of the integrity of the viral particles.
A number of international efforts have been attempted in order to standardize the method but there is as yet neither a harmonized protocol nor an international FMDV standard. The technical complexities of the method and the requirement of specialized items of equipment have probably contributed to this situation (Barteling S J. Need for further standardization of the 146-S test as the basis for final-Foot-and-Mouth Disease (FMD) Vaccine formulation. EUFMD Research Group Meeting 1999—Appendix 17).
The lack of a standardized method to measure the active ingredient present in FMD vaccines is an important factor to explain why expensive and cumbersome clinical trials, typically involving 17 to 20 large animals (the target species) per batch, are still required for registration of FMD vaccines and batch release.
Over the last decades, human and veterinarian health industries have witnessed increasingly stringent government regulations related to Good Manufacturing Practice (GMP) for the production of pharmaceutical and biopharmaceuticals products. New quality concepts have arisen, like Process Analytical Technology (PAT), which Food and Drug Administration (FDA) defines as a mechanism to design, analyze, and control pharmaceutical manufacturing processes through the measurement of Critical Process Parameters (CPP) that affect Critical Quality Attributes (CQA). PAT emphasizes the importance of controlling the production process as a means to achieve the highest quality standard for the final product.
For the vaccine industry and especially for vaccines based on full-size antigens (like inactivated viral vaccines or live attenuated viral vaccines), methods and equipment capable of reliably quantifying and determining the size of particles in the nanometer (nm) range would represent important tools for the implementation of in-process controls meant to ensure the quality of the viral antigens produced at every step of the process. Nevertheless, the analysis of complex process streams containing virus particles is a much more difficult task than the analysis of common recombinant proteins like monoclonal antibodies (mAbs). One challenge is that manufacturing practices for many current vaccines were developed decades ago, before the rise of serum-free media culture technology. The serum containing medium used in many manufacturing processes contains a very high content of proteins. Also, the yield of virus particles obtained from the infection of cell culture is generally in the order of milligram per liters, which is several orders of magnitude lower compared to the expression of recombinant proteins such as mAbs. Also of great importance is the fact that some viruses, like FMDV, have lytic replication cycle that triggers the destruction of the cell. The release in the cell culture medium of all the materials contained in the cytoplasm and the nucleus of the cells, including all the genomic material and lipid residues from the cell membrane will make the analysis more difficult.
Since size exclusion chromatography separates different molecules based on their hydrodynamic volumes, molecules of different molecular weights but similar hydrodynamic volumes are prone to show very similar mobility behavior during chromatography runs. In the conditions of very complex process streams, size exclusion chromatography is usually unable to provide a resolution good enough in order to separate the peak of virus particles of interest from other process contaminants of similar hydrodynamic volume such as large molecules of DNA and large lipid residues.
Protein or viral-based vaccines are vaccine models usually employed for delivering antigens to a subject for immunization. It is extremely crucial that the antigen being delivered is of its correct or native conformation such that appropriate epitopes can be presented to induce specific and effective immune responses. Therefore, misfolded, degraded or aggregated antigen may have reduced efficacy in, or even not be capable of, triggering specific immune responses. Dynamic Light Scattering (DLS) helps to assess the efficacy of protein or viral-based vaccines in terms of their integrity and stability. By analyzing the DLS profile, one would be able to check whether the antigens have been degraded or aggregated over time or upon storage in a particular buffer or temperature, and thereby assuring the efficacy of the vaccines.
Separation methodologies such as size-exclusion chromatography, when used alone, usually give rough estimates of particle size by close scrutiny of mobility behavior but these assessments are not sensitive enough to detect subtle modifications of antigen dimension. In contrast, DLS sensitively and accurately detects and estimates size of particles, and hence allows monitoring subtle differences of the particles' size. However, DLS can only analyze the integrity and estimate the size of particles with high purity.
Due to the economic relevance of FMD worldwide, there is a continuous need of improvements in the prevention tools, particularly regarding improved vaccines and/or methods of preparation thereof. The present invention provides methods for the quantification and characterization of FMDV and related products in a high-throughput and accurate manner. With the growing demand for both quantity and quality of FMDV vaccines, the present invention is a very useful and cost-effective tool for the quality control of the FMDV vaccines.
As an example, the world biggest FMD vaccine market, the Chinese market, requires 1.7 billion doses (of 2 milliliters each) per year. Considering that one average industrial batch of vaccine represents 5 million doses (i.e., a 10000 liters batch), this means that around 340 batches of FMD vaccines are produced each year in China by different manufacturers. As of today and because of this huge number of batches produced, the Chinese Veterinary Regulatory Authority has forfeited the responsibility of controlling the quality of the FMD vaccine batches and has to rely on the performance of quality control tests by each vaccine manufacturer. The quality control of FMD vaccines is usually monitored using in vivo potency testing which allows assessment of the quality of the vaccines in bulk quantity but requires complex and cumbersome procedures. Currently, there are no other in vitro techniques available that would enable the quality control of such a great quantity of batches of vaccine in a timely manner.
The present invention, for the first time, introduces an in vitro system that is capable of quantifying the antigen payload per dose and characterizing the size and the integrity of the inactivated antigen in each vaccine batch, in a high throughput manner. The present invention implements a much more simplified system than the in vivo potency testing system for the quality control of the FMD vaccines and thereby improves the quality of the products. It is estimated that the present invention can monitor the quality of 340 batches of vaccines in just a few days.
Thus, the present invention would eventually guarantee a better quality of the vaccines and confer a better protection against the Food and Mouth Disease to the animal population.